Zootaxa 2032: 1–38 (2009) ISSN 1175-5326 (print edition) www.mapress.com/zootaxa/ Article ZOOTAXA Copyright © 2009 · Magnolia Press ISSN 1175-5334 (online edition)
Integrative taxonomy identifies new (and old) species in the Lasioglossum (Dialictus) tegulare (Robertson) species group (Hymenoptera, Halictidae)
JASON GIBBS York University, Department of Biology, 4700 Keele St., Toronto, Ontario, M3J1P3, Canada. E-mail: [email protected]
Table Of Contents
Abstract ...... 1 Introduction ...... 2 Materials and methods ...... 3 Results ...... 5 Lasioglossum (Dialictus) tegulare species group...... 13 Lasioglossum (Dialictus) tegulare (Robertson), comb. n...... 13 Lasioglossum (Dialictus) ellisiae (Sandhouse), comb. n...... 18 Lasioglossum (Dialictus) lepidii (Graenicher), comb. n...... 22 Lasioglossum (Dialictus) puteulanum Gibbs, sp. n...... 25 Lasioglossum (Dialictus) carlinvillense Gibbs, sp. n...... 28 Key to eastern species ...... 32 Discussion ...... 32 Acknowledgements ...... 34 Literature cited ...... 35
Abstract
An integrative taxonomic approach that utilizes the DNA barcode region of cytochrome c oxidase subunit 1 in conjunction with traditional morphological approaches identifies five distinct species previously recognized as Lasioglossum (Dialictus) tegulare (Robertson). Differences in DNA sequences and congruent, albeit minor, morphological variation support separation of L. tegulare into five species. Unique nucleotide substitution patterns for each species allows for character-based diagnostics using DNA barcodes. The names L. ellisiae (Sandhouse) and L. lepidii (Graenicher) are removed from synonymy. Two new species, L. puteulanum Gibbs sp. n. and L. carlinvillense Gibbs sp. n., are described. A key is provided, which permits the identification of both males and females. The utility of the DNA barcode region as part of an integrative taxonomic framework is discussed.
Key words: Cryptic species, integrative taxonomy, DNA barcodes, Lasioglossum, Dialictus, Halictidae
Accepted by E. Almeida: 20 Feb. 2009; published: 11 Mar. 2009 1 Introduction
Several methods for automated species identification using quantitative approaches have been proposed (Gaston & O’Neill 2004) but none has garnered the attention of DNA barcoding. DNA barcoding is a new method that promises to speed taxonomic progress and allow identification of specimens even without taxonomic expertise (Hebert et al. 2003a). DNA barcoding employs a short strand of a standard gene to identify species. A 657-bp fragment of the mitochondrial cytochrome c oxidase subunit I (COI) is the chosen standard for animals (Hebert et al. 2003a). DNA barcoding has incited much controversy in the taxonomic community and has been both lauded and denounced in the literature (e.g. Trewick 2008 and references therein). Integrative taxonomic approaches that combine morphological, molecular and other types of data are the best methods for describing species (Dayrat 2005; Page et al. 2005; DeSalle et al. 2005). Morphological and molecular data have complementary strengths (Hillis 1987; Hillis & Wiens 2000) and in combination can overcome weaknesses of single datasets alone (e.g. Wahlberg et al. 2005). Molecular evidence can provide an independent test of morphological assessments of species identity and vice versa (Page et al. 2005). Multiple sources of data (e.g. morphology, DNA, geography) are needed to test and corroborate hypothetical species limits (DeSalle et al. 2005). DNA barcoding efforts provide molecular data that may aid in discovering cryptic species (Hebert et al. 2004; Yassin et al. 2007) but these findings should be incorporated into an integrative taxonomic framework (Dayrat 2005; DeSalle et al. 2005). Confounding factors such as incomplete lineage sorting or diversifying selection acting on morphological traits can result in closely related species that cannot be differentiated by a single piece of molecular evidence (Avise 2000; Funk & Omland 2003). In this respect, the DNA barcode region does not differ from other candidate genes. DNA barcoding efforts are novel relative to other molecular methods by virtue of the standardization and taxonomic breadth for which DNA sequence data is being made available. One advantage of selecting the DNA barcode region for integrative taxonomic purposes over other genes is that the data can be used both for alpha taxonomy and for species identification. The importance of bees as pollinators (Buchmann & Nabhan 1996; Klein et al. 2007) and their potential as ecosystem monitors (Zayed et al. 2004) makes their study of particular importance. The large number of bee species (>19,000 valid species-names worldwide [Ascher et al. 2008]) with many highly speciose genera and subgenera makes species recognition difficult. In many cases, cryptic species, caste differentiation and sexual dimorphism add to the puzzle (Sandhouse 1924; Knerer & Atwood 1962; Janjic & Packer 2001; Pilgrim & Pitts 2006; Sheffield & Westby 2007). Molecular evidence, such as DNA barcodes, may not only differentiate cryptic species (Carman & Packer 1997; Packer & Taylor 1997; Danforth et al. 1998; Hebert et al. 2004; Witt et al. 2006; see Avise 2000, 2004 for further examples) but also associate queens, workers, larval stages and dimorphic sexes (Pilgrim & Pitts 2006; Gibbs in press) that would otherwise be misidentified (or not identified further than subgenus) based on morphology alone. The subgenus Dialictus (Halictidae: Lasioglossum) is one of the most taxonomically difficult groups amongst the bees. In North America, Dialictus are both speciose (over 270 currently recognized names) and the most commonly collected subgenus of bee (MacKay & Knerer 1979; Eickwort 1988; Grixti & Packer 2006; Campbell et al. 2007). Dialictus are also notoriously difficult to identify to species because they are “morphologically monotonous” (Packer 1997; Michener 2007). In most cases, only very subtle differences can be used to differentiate closely related species. Identification is further complicated by the existence of castes in the many eusocial species (reviewed in Michener 1974; Packer 1993; Yanega 1997). In bee diversity studies many Dialictus cannot be identified to the species level (e.g. Giles & Ascher 2006) preventing more in depth study of sociobiology, biodiversity and pollination biology. The extreme similarity between species may be due in part to a recent origin and rapid diversification of the speciose lineage containing Dialictus, likely to have started 20–22 million years ago (Brady et al. 2006). Very little taxonomic progress has been made on this group in the last forty years (Mitchell 1960; Knerer & Atwood 1966), and there is much work to be done. The nominal species Lasioglossum tegulare (Robertson), widespread in eastern North America, is one of
2 · Zootaxa 2032 © 2009 Magnolia Press GIBBS the few currently recognized species of Dialictus that is easy to identify. As the name suggests, this species has a distinctive tegula (Fig. 1), which is remarkable for its size, shape and punctation. Two additional species from the eastern United States, L. marinum (Crawford) and L. surianae (Mitchell), have similar tegulae but are easily distinguished morphologically from L. tegulare. The microsculpture of L. tegulare, as well as its size and colouration, differ from L. marinum and the two are not close relatives (Gibbs, unpublished data). Lasioglossum surianae has distinct colouration patterns that easily distinguish it from L. tegulare. The presence of the elongate tegula was not mentioned in Mitchell’s (1960) key or description of L. surianae but inspection of the holotype clearly shows this character. Lasioglossum marinum is a sand dune specialist uncommonly collected outside of coastal areas from Florida to Massachusetts (Graenicher 1927; Moure & Hurd 1987), while L. surianae is only known from the Florida Keys (Mitchell 1960) and the Bahamas. The L. tegulare species group extends into South America but will require considerable additional study. Two other species with large tegulae, L. ellisiae (Sandhouse) and L. lepidii (Graenicher), have been described from eastern North America but were synonymized with L. tegulare (Mitchell 1960). Easily recognized species may not be examined in as great detail as taxa known to belong to difficult species complexes and may be a repository for cryptic species (Packer & Taylor 1997). I report the existence of cryptic species within the “species” L. tegulare identified through reciprocal illumination from DNA barcodes and morphology. Previous DNA barcode studies have usually used phenetic methods (Prendini 2005) and left putative new species for future workers to describe (e.g. Hebert et al. 2004). In this study I apply more phylogenetically rigorous parsimony and Bayesian methods to study patterns in the molecular evidence. I also provide detailed morphological description of the new species. The taxonomic limits of L. tegulare are revised with the description of two new cryptic species, L. puteulanum Gibbs sp. n. and L. carlinvillense Gibbs sp. n. and the removal of the names L. ellisiae and L. lepidii from synonymy.
Materials and methods
DNA extraction, PCR and sequencing were done at the Canadian Centre for DNA Barcoding at Guelph University (Guelph, Ontario) using standard protocols described elsewhere (Hebert et al. 2003b) and available online at http://www.dnabarcoding.ca/pa/ge/research/protocols. Universal primers for amplifying the DNA barcode sequence for insects (LCO1490 and HCO2198; Folmer et al. 2004 or the variants LEPF and LEPR; Hebert et al. 2004) were used. DNA barcodes were generated from L. tegulare specimens throughout its range (ON to FL). Two alternate outgroups were used, L. (Hemihalictus) lustrans (Cockerell) and L. (Evylaeus) quebecense (Crawford) based on a higher level phylogeny for Lasioglossum (Danforth et al. 2003). The outgroups belong to the acarinate Evylaeus and carinate Evylaeus groups respectively (Danforth et al. 2003). Lasioglossum marinum and L. surianae were included to compare the relationships of the L. tegulare species group to other eastern species with enlarged tegulae. Sequences from L. vierecki were included based on the results of previous studies (Danforth et al. 2003), wherein L. vierecki appears in the sister clade of the tegulare species group. Only those sequences that were greater than 600bp with at most one ambiguous base pair were included in the phylogenetic analysis. DNA barcode sequences were uploaded to BOLD: the barcode of life data system (Ratnasingham & Hebert 2007). The analytical system within BOLD was used to generate a neighbour-joining (NJ) tree based on a Kimura-2 parameter model of base substitution (Kimura 1980). To determine if the patterns observed in the NJ tree were robust to more rigorous methods, sequences were input into TNT (Goloboff et al. 2003a) for parsimony-based phylogenetic analyses. A driven search was performed using default settings with the following exceptions: ratchet (200 iterations, up and down weight probabilities set to 10), drift (20 cycles) and ‘find minimum length’ set to 100. Symmetric resampling (Goloboff et al. 2003b) was performed on the results using groups from the tree with a 33% probability of changing the weights (up or down) for 10000 replicates. Support values are indicated on the tree using ratio of groups supported or contradicted (GC). For each node, the difference between the frequency of the group and
LASIOGLOSSUM (DIALICTUS) TEGULARE SPECIES GROUP Zootaxa 2032 © 2009 Magnolia Press · 3 FIGURE 1. Dorsal views of enlarged and punctate tegulae from a L. tegulare species group (A) female, (B) male. (C) A tegula from a typical L. (Dialictus) species.
4 · Zootaxa 2032 © 2009 Magnolia Press GIBBS the most frequent alternate arrangement is calculated. GC values range from -100 to +100, indicating maximum contradiction (alternate arrangement is found in all resampled matrices) to maximum support (group found in all resampled matrices). A Bayesian phylogenetic analysis was performed using MrBayes v. 3.1.2 (Huelsenbeck & Ronquist 2001) on the DNA barcode dataset. The data were partitioned by codon position. The general time reversible model (GTR) of evolution was applied with the rate of evolutionary change based on a gamma distribution. Four chains were run simultaneously. The analysis was continued until the standard deviation of split frequencies decreased to a suitable level (below 0.01). In total, 2,000,000 generations were run with trees sampled from every fiftieth generation for a total of 40,000 trees. The unstable “burn-in” region was removed by deleting the initial 25% of trees in the analysis. Species descriptions are included for the cryptic species identified using integrative taxonomy. Descriptions follow the format used in Toro and Moldenke (1979) and are subdivided into sections on colouration, pubescence, surface sculpture and structure. The subsections themselves proceed from the anterior to posterior of the specimen. Terminology for structures and surface sculpture largely follow Michener (2007) and Harris (1979), respectively. Additional terminology related to the propodeum is based on Murao and Tadauchi (2007) as follows: The term ‘lateral slope’ refers to the dorsal surface of the propodeum laterad of the metapostnotum. The term ‘oblique carina’ refers to the carina that separates the lateral slope from the posterior surface. The appressed hairs on the declivous surface of the first tergum, when present, are referred to as the acarinarial fan. Measurements and abbreviations follow Gibbs and Packer (2006). The following abbreviations are used: IOC—interocellar distance, OOC—ocellocular distance, UOD —upper ocular distance, LOD—lower ocular distance, i—interspace, d—puncture diameter (these are used in conjunction to give a relative measure of puncture density), and OD—median ocellar diameter (this abbreviation is primarily used to give a relative measure of hair length). The OD for each species is approximately 0.12 mm. Individual terga, sterna and flagellomeres are referred to by the letter T, S, and F, respectively, followed by the appropriate number. Relative size measurements are given in ratios based on eyepiece graticule units. This is meant to prevent the use of unwieldy measurements in standard units. Ratios based on measurements of individual body parts were taken from the type specimen. Head, body and forewing length and head breadth were measured from additional specimens but values from the holotype are given before parentheses. A dichotomous key is included. The following abbreviations are used for institutions in which specimens examined are deposited: ANSP: Academy of Natural Sciences, Philadelphia, PA; CUIC: Cornell University Insect Collection, Ithaca, NY; INHS: Illinois Natural History Survey, Champaign, IL; SEMC: Kansas University Natural History Museum (Snow Entomological Collection), Lawrence, KS; NMNH: National Museum of Natural History, Washington, DC; PCYU: Packer’s Apoidea Collection at York University, Toronto, ON; UWGB: University of Wisconsin, Green Bay (Richter Museum of Natural History), WI. DNA barcoded specimens used in the phylogenetic analysis are annotated as such and for new species are designated as paratypes.
Results
The DNA barcode region of COI was sequenced from specimens identified based on morphology as Lasioglossum tegulare. Unexpectedly high levels of sequence divergence were noted among specimens of L. tegulare. A more comprehensive study of morphology and more thorough sampling of DNA barcode sequences provided evidence of a species complex previously misinterpreted as a single species. I obtained seventy-one DNA barcode sequences of lengths 604–658bp, with no more than a single ambiguous base pair per sequence. All sequences are publicly available on BOLD (LTEG project) and GenBank (accession numbers FJ663058–FJ663128) (Table 1). All sequences from the L. tegulare species complex lacked indels, in-frame stop codons, non-synonymous mutations and each had a highly similar GC% (25.18, SE = 0.053, min = 24.06, max = 25.9) with an extreme AT-bias in the third codon position (95.43, SE
LASIOGLOSSUM (DIALICTUS) TEGULARE SPECIES GROUP Zootaxa 2032 © 2009 Magnolia Press · 5 = 0.1, min = 94.05, max = 96.69). These characteristics are inconsistent with amplification of nuclear pseudogenes (Song et al. 2008).
TABLE 1. Lasioglossum species included in this study with BOLD process ID and GenBank accession numbers. Numbers in brackets indicate ambiguous base pairs. Subgenus Species BOLD Genbank Length State or Process ID Accession Numbers (bp) Province Evylaeus quebecense DLII816-07 FJ663099 658 VA Hemihalictus lustrans DIAL511-06 FJ663075 658 NC Dialictus marinum DIAL746-06 FJ663076 614 VA DIAL747-06 FJ663077 658 VA DIAL748-06 FJ663078 658 VA DIAL749-06 FJ663079 658 VA DIAL763-06 FJ663080 658 VA DLII418-07 FJ663081 645 VA DLII685-07 FJ663082 657 MD vierecki DIAL033-06 FJ663128 619 MD DIAL034-06 FJ663127 624 MD DIAL035-06 FJ663119 619 MN DIAL121-06 FJ663120 658 MB DIAL358-06 FJ663121 614 IN DIAL370-06 FJ663122 614 NY DIAL479-06 FJ663123 622 NC DIAL480-06 FJ663124 658 SC DIAL484-06 FJ663125 658 SC DIAL915-06 FJ663126 654 ON surianae BEECB173-07 FJ663100 632 Bahamas tegulare DIAL091-06 FJ663102 658 VA DIAL366-06 FJ663109 614 NY DIAL367-06 FJ663110 614 VA DIAL485-06 FJ663111 658 WV DIAL490-06 FJ663112 658 WV DIAL493-06 FJ663113 658[1] WV DIAL495-06 FJ663114 658 NC DIAL518-06 FJ663115 658 SC DIAL683-06 FJ663103 658 DC DIAL684-06 FJ663104 658 DC DIAL742-06 FJ663105 614 VA DIAL743-06 FJ663106 658 VA DIAL744-06 FJ663107 618 VA DIAL745-06 FJ663108 604 VA DIAL824-06 FJ663116 653 VA to be continued.
6 · Zootaxa 2032 © 2009 Magnolia Press GIBBS TABLE 1. (continued) Subgenus Species BOLD Genbank Length State or Process ID Accession Numbers (bp) Province DIAL1050-06 FJ663117 658 SC DIAL1051-06 FJ663118 658 SC DLII468-07 FJ663101 645 MD ellisiae BEECB213-07 FJ663069 658 MN BEECB221-07 FJ663068 658 MN DIAL356-06 FJ663063 586 IN DIAL476-06 FJ663064 658 IN DIAL653-06 FJ663061 658 ON DIAL654-06 FJ663062 658 ON DIAL888-06 FJ663065 658 NC DIAL918-06 FJ663067 654 ON DIAL1089-06 FJ663066 658 NC DLII031-06 FJ663070 645 ON DLII833-07 FJ663071 658 MA lepidii DIAL863-06 FJ663072 655 FL DIAL865-06 FJ663073 658 FL DIAL867-06 FJ663074 658 FL puteulanum DIAL027-06 FJ663083 614 FL DIAL028-06 FJ663084 615 FL DIAL029-06 FJ663085 615 FL DIAL473-06 FJ663086 658[1] FL DIAL487-06 FJ663087 658[1] NC DIAL496-06 FJ663088 644 SC DIAL498-06 FJ663089 658 SC DIAL501-06 FJ663090 658[1] SC DIAL513-06 FJ663091 658[1] FL DIAL514-06 FJ663092 658[1] FL DIAL866-06 FJ663093 622 FL DLII435-07 FJ663094 645 SC DLII465-07 FJ663095 619 SC DLII472-07 FJ663096 635 FL DLII474-07 FJ663097 619 FL DLII696-07 FJ663098 611 FL carlinvillense DIAL728-06 FJ663058 658 IL DIAL832-06 FJ663059 636 IL DIAL837-06 FJ663060 658 IL
A neighbour-joining tree of the seventy barcode sequences shows deep divergences within the nominal species L. tegulare (Fig. 2). The five putative species, previously known as L. tegulare, each form a distinct
LASIOGLOSSUM (DIALICTUS) TEGULARE SPECIES GROUP Zootaxa 2032 © 2009 Magnolia Press · 7 cluster within the tree. Pair-wise sequence divergences between species are summarized in Table 2. The average pair-wise sequence divergence among the five species was 3.06%. Lasioglossum puteulanum and L. tegulare were found to have the most similar DNA barcodes. The minimum sequence divergence between this species pair was only 1.7%. The maximum intraspecific divergence was 0.9%, slightly more than half (0.53) the minimum interspecific value. A clear distinction can then be made between intra- and interspecific variation.
FIGURE 2. Neighbour-joining tree based on DNA barcode sequences implemented using the analytical tools in BOLD with L. lustrans as the outgroup. Individual sequences are marked with the standard two letter abbreviation for the province or state of origin.
The parsimony analyses for the two alternative outgroups, L. lustrans and L. quebecense, found 525 and 268 equally most parsimonious trees of length 238 and 255 steps, respectively, after sampling more than 11 billion rearrangements each. A strict consensus of the equally most parsimonious trees from each analysis
8 · Zootaxa 2032 © 2009 Magnolia Press GIBBS collapsed many nodes but each putative species resolved as a distinct monophyletic unit (Fig. 3). The strict consensus trees of the two parsimony analyses differed in only one node of the L. vierecki clade that was collapsed or resolved when the outgroup was L. lustrans or L. quebecense, respectively. Symmetric resampling showed very strong support (GC value >95 or >93) for all putative species except L. puteulanum (GC value 52 or 67).
FIGURE 3. Strict consensus of 525 equally most parsimonious trees (length = 238) found with L. lustrans set as the outgroup. Numbers at nodes indicate GC values (Goloboff et al., 2003b) greater than 50 after 10000 replicates using default setting traditional searches with TNT for each replicate (Goloboff et al., 2003a). Upper and lower GC values are from analyses with the outgroup as L. lustrans and L. quebecense, respectively.
LASIOGLOSSUM (DIALICTUS) TEGULARE SPECIES GROUP Zootaxa 2032 © 2009 Magnolia Press · 9 FIGURE 4. Most probable tree based on Bayesian analysis using L. lustrans as the outgroup. Numbers at basal node of each species indicate posterior probabilities. Upper and lower probabilities are from analyses with the outgroup as L. lustrans and L. quebecense, respectively.
TABLE 2. Summary of pair-wise sequence differences. Upper triangle indicates minimum percent pair-wise sequence divergence between species. Numbers on diagonal indicate maximum intraspecific sequence diversity. Lower triangle indicates number of sites that do not share nucleotides in common between species pairs. Species L. tegulare L. ellisiae L. lepidii L. puteulanum L. carlinvillense L. tegulare 0.9 3.9 3.4 1.7 2.7 L. ellisiae 22 0.8 3.6 3.6 3.5 L. lepidii 17 22 0.6 3.3 2.3 L. puteulanum 8 17 17 0.8 2.6 L. carlinvillense 14 22 14 15 0.0
10 · Zootaxa 2032 © 2009 Magnolia Press GIBBS TABLE 3. Invariant nucleotides not shared by other members of the L. tegulare species complex. Nucleotide position is given relative to the full length COI gene of Apis mellifera. Nucleotides present in other species of the complex (often invariant) are given in order of frequency. Species # of unique Nucleotide Position Invariant nucleotide Most common nucleotide in substitutions (relative to Apis) other species L. tegulare 2 159 C T 258 T A,G L. ellisiae 7 75 T A,G 210 T A 324 C T 354 A T 357 A T 558 T A 615 C T L. lepidii 4 414 A T,C 594 G A 636 C T 666 G A L. puteulanum 2 96 A T 381 C T L. carlinvillense 3 255 C T 414 C T,A 621 C T L. surianae 13 57 A T 147 C T 162 C T 165 C T 318 C A 330 G A 363 G A 400 C T 441 C A 459 C T 465 C T 516 C T 537 C T 606 T A
The results of the Bayesian analyses are largely congruent with the parsimony trees. Each of the putative species forms a distinct monophyletic group with high posterior probability (99–100) for all putative species except L. puteulanum (76) (Fig. 4). One important difference between the Bayesian trees and the previous
LASIOGLOSSUM (DIALICTUS) TEGULARE SPECIES GROUP Zootaxa 2032 © 2009 Magnolia Press · 11 results is the relative position of the L. puteulanum clade. In both the NJ and parsimony trees, L. puteulanum is sister group to L. tegulare s. str. (Figs. 2, 3). In contrast, L. puteulanum appears as a basal branch and sister group to the remaining species in the complex in the Bayesian result (Fig. 4). The node supporting the joint L. tegulare s. str. and L. puteulanum clade had low support (GC value 38 or 49) in the parsimony tree.
FIGURE 5. Distribution map of L. tegulare species group east of the Mississippi (excluding L. surianae). Open squares = L. tegulare; black circles = L. ellisiae; black triangles = L. lepidii; open circles = L. puteulanum; open triangles = L. lepidii.
The tree topologies among the various analyses are all largely congruent with some minor variation among the within species relationships. All analyses agree that the putative species are monophyletic. Choice
12 · Zootaxa 2032 © 2009 Magnolia Press GIBBS of outgroup had no effect on the species-level topology. The relative positions of L. marinum and L. vierecki in each analysis support the hypothesis that L. marinum does not belong to the L. tegulare species group and evolved an elongate tegula independently. The position of the L. surianae sequence in each analysis renders the L. tegulare s. l. sequences paraphyletic. The paraphyletic pattern of the DNA barcode sequences from the nominal species L. tegulare and the congruent pattern of morphology and DNA are consistent with the presence of cryptic species in L. tegulare (Funk & Omland 2003). Each of the five species (and also L. surianae) has unique invariant nucleotide substitutions not shared by other members of the L. tegulare species complex (Table 3). The number of unique substitutions ranges from two to seven among the five species examined here. These substitutions could be used as diagnostic characters for identifying these species. The possibility of DNA-based identification of these species is therefore not limited to distance-based methods. Morphological comparison of the five putative cryptic species and L. surianae found subtle differences among individuals belonging to different clades in the DNA barcode-phylogenies. Proper association of dimorphic sexes was possible by virtue of the DNA barcode data. Division of L. tegulare into multiple species is justified by the analysis of the DNA sequence data and morphological study. Partial corroboration also comes from the geographical distributions of the species that occupy incompletely overlapping ranges (Fig. 5). By comparison to type material, it was found that two of these species correspond with the previously synonymised names L. ellisiae and L. lepidii. The remaining cryptic species are described herein.
Lasioglossum (Dialictus) tegulare species group
Diagnosis. Members of the L. tegulare group can be distinguished from most other Dialictus by the presence of an enlarged and punctate tegula. In North America, there are two exceptions; L. marinum and L. megastictum (Cockerell) both have enlarged tegula but additional morphological data do not support a close relationship to L. tegulare (Gibbs, unpublished data). Both of these latter species are larger than members of the tegulare group and have dense punctation on the mesoscutum. In contrast, species within the tegulare group are small to at most medium-sized Dialictus and have close but distinctly separated punctures on the central disc of the mesoscutum. In addition, L. marinum has metallic reflections on the metasoma that are absent in the tegulare group. Lasioglossum megastictum lacks punctures on the mesepisternum that are a common feature of many tegulare group species. Description. Small to medium sized (3.5–6.1 mm); andreniform; head and mesosoma dull metallic; metasoma dark brown to piceous, rarely ferruginous (as in L. hunteri [Crawford]); mesoscutum punctation close but distinctly separated on centre of disc; mesepisternum often distinctly punctate; tegula enlarged forming posterior angle often coarsely punctate; basal vein arched; 2nd and 3rd submarginal crossveins (1rs-m and 2rs-m) and 2nd recurrent vein (2m-cu) weak.
Lasioglossum (Dialictus) tegulare (Robertson), comb. n. (Figures 6A–D, 7A–C)
Halictus tegularis Robertson, 1890: 318. ♀ ♂. Chloralictus tegularis: Robertson, 1902: 248 (key). Halictus (Chloralictus) tegularis: Viereck, 1916: 706 (key). Lasioglossum (Chloralictus) tegulare: Michener, 1951: 1118 (catalogue). Dialictus tegularis Mitchell, 1960: 423 (♀ ♂ redescriptions).
Diagnosis. Females of L. tegulare can be recognized by the following combination of characters: head and mesosoma pale to golden green, paraocular area with sparse, subappressed hairs which do not obscure the surface, distinct microsculpture between punctures of mesoscutum and mesepisternum, and three or four teeth
LASIOGLOSSUM (DIALICTUS) TEGULARE SPECIES GROUP Zootaxa 2032 © 2009 Magnolia Press · 13 on the inner hind tibial spur (not including apex of rachis). Females of L. puteulanum have the head and mesosoma deep blue. Females of L. lepidii have dense subappressed hairs on the paraocular area which obscure the surface adjacent to the inner eye margin and lower paraocular area. Females of L. ellisiae have the integument of the mesoscutum (particularly adjacent to parapsidal lines) and mesepisternum smooth, with at most faint microsculpture which gives these areas a shiny appearance. Females of L. carlinvillense have only two teeth on the inner hind tibial spur. Males of L. tegulare can be recognized by the following combination of characters: ventral surface of flagellum pale, appressed hairs of face mostly limited to paraocular areas and not obscuring clypeus, and T2–T3 punctures uniformly dense on disc basal to the premarginal line. Males of L. ellisiae have the ventral surface of the flagellum dark to ferruginous and T2–T3 punctures dense on basal half but sparse approaching premarginal line. Males of L. lepidii have the ventral surface of the flagellum bright yellow and appressed hairs of the face dense, obscuring the majority of the clypeus. Males of L. puteulanum have appressed hairs more uniformly distributed on face with less than half of the clypeal surface obscured. Redescription. Female. Length: 4.7 (4.0–4.9) mm, fore wing length: 3.3 (2.8–3.5) mm head width: 1.4 (1.2–1.4) mm, head length: 1.3 (1.1–1.3) mm, n=20 Colouration. Head and mesosoma dull metallic pale green except the following: labrum brown-piceous; mandible base brown, apex red; clypeus brown, golden metallic above; supraclypeal area lighter green to golden; antennae brown, ventral surface of flagellomeres ruddy-brown except F8–F10 ventral surface testaceous; mesoscutum green to golden green; tegula piceous with central area ferruginous; legs brown, fore medio- and distitarsi reddish, apex of mid and hind distitarsi testaceous; wing venation and pterostigma testaceous-brown; wings faintly dusky; propodeum with green to blue reflections; metasoma piceous-brown; marginal zones of terga and sterna light brown. Pubescence. Lower paraocular area with sparse, subappressed, plumose hairs; head and mesosoma with sparse, erect, plumose hairs (1–1.5OD), longer on metanotum and ventral surface of mesosoma (2OD); posterolateral margin of pronotum and pronotal lobe with dense, appressed tomentum; dense scopa on hind femur; propodeal lateral surface hairs (2OD) with long branches; acarinarial appressed fan complete; terga with sparse, erect hairs (1–2OD), more abundant on ventrolaterally reflexed portions; T3–T5 ventrolaterally reflexed areas with few erect hairs longer (2.5–3OD); T2–T3 basolateral areas and T4 dorsal surface with sparse appressed, plumose hairs; sterna with long, posteriorly oriented hairs emerging from apical half of disc (2–3OD); S1–S4 hairs with long branches. Surface sculpture. Clypeus glabrate except upper margin imbricate, punctures moderately coarse (i=1–2d), fine above (i=d); supraclypeal area smooth and shining below, imbricate above, punctures fine (i=1–1.5d); lower paraocular area imbricate, glabrate below, punctures moderately coarse and deep (i
14 · Zootaxa 2032 © 2009 Magnolia Press GIBBS surface of propodeum (1.8:1.0:1.4); tegula elongate, angulate posteromedially; inner hind tibial spur pectinate with three subapical teeth; oblique propodeal carina weakly evident, not contiguous with lateral carina.
FIGURE 6. Lasioglossum tegulare A) face of female holotype; B) lateral habitus of female holotype; C) face of male; D) lateral habitus of male.
Male. Length: 3.8 mm, fore wing length: 2.75 mm, head length: 1.1 mm, head width: 1.1 mm Colouration. Head and mesosoma dull metallic green with faint golden reflections except gena with blue reflections; mesepisternum bluish-green below; propodeum bluish; the following parts dark brown-piceous: labrum; mandible except apex reddened; lower clypeus; antenna, lighter basally, F1–F3 testaceous ventrally; tegula; wing venation and pterostigma brown; wings hyaline, apex faintly dusky; metasoma. Pubescence. Inter-antennal area and paraocular area from mandible base to emarginated portion of eye with subappressed hairs; head, mesosoma, anteriorly directed surface of T1 and ventrolaterally reflexed portions of terga with sparse, erect hairs (1.5–2OD); T1–T4 with short laterally oriented setae; sterna with erect hairs, densest on S4–S5. Surface sculpture. Head and mesosoma glabrate and shining; clypeus (i LASIOGLOSSUM (DIALICTUS) TEGULARE SPECIES GROUP Zootaxa 2032 © 2009 Magnolia Press · 15 mesepisternum punctures moderately coarse and deep (i=d); metapostnotum irregularly striate, laterally striations extending onto anterior half of lateral slope; lateral and posterior propodeal surfaces shiny-imbricate with deep, moderately fine punctures (i=1–1.5d); terga smooth, very faintly coriarious; terga punctures fine but deep and distinct (i=1–1.5d), apically impressed areas impunctate; anteriorly directed surface of T1 largely impunctate. Structure. Face length subequal to breadth; eyes convergent below (UOD:LOD = 1.5:1.0); clypeus protruding slightly more than one half below lower ocular tangent; antennal sockets slightly nearer to each other than to inner eye margin; frontal line carinate ending less than 2OD from median ocellus; OOC less than IOC; eye wider than gena from lateral view; hypostomal carinae parallel; pedicel subequal in length to F1; F2–F10 length 1.5 times breadth, F11 longer; ratio of lengths of mesoscutellum: metanotum: dorsal surface of propodeum (1.9:1.0:1.6); tegula enlarged, posterior margin not strongly angled; propodeal carina not evident between dorsal and posterior surfaces; metasoma narrow relative to female. Terminalia. Median lobe of S7 narrow and elongate, sides subparallel, apex rounded (Fig. 7A); apical margin of S8 weakly convex (Fig. 7B); genitalia as shown in Fig. 7C; gonostylus with few erect dorsal hairs near base; retrorse lobes elongate, attenuated apically, covered with fine setae. Range. Vermont to South Carolina, west to Ontario, Kentucky and Tennessee. Specimens examined. Holotype, USA, CONNECTICUT: HOLOTYPE ♀, N. Haven, 6.vi.1878, (WH Patton) (ANSP); RHODE ISLAND: 2 ♀, Newport Co., N41.4969 W71.3678, 22.vii.2005, (P Ostenton); MASSACHUSETTS: 1 ♀, Middlesex Co., Harvard:Oxbow NWR, Wallace Rd., 28.v.2006, (MF Veit); 1 ♀, Middlesex Co., Dunstable, sandpit 0.1mi E of airport, 29.iv.2006, (MF Veit) (PCYU); VERMONT: Windham Co., N42.4969 W71.3678, 22.vii.2005, (P Ostenton) (PCYU); NEW YORK: 1 ♀, Suffolk Co., 6.ix.2005, (SW Droege) [Barcoded]; 1 ♀, Suffolk Co., N41.13403 W72.3247, 8.ix.2005, (SW Droege); 1 ♀, Suffolk Co., 8.ix.2005, (SW Droege); 1 ♀, Suffolk Co., N41.05132 W71.9519, 7.ix.2005, (SW Droege); 2 ♀, Suffolk Co., 6.ix.2005, (SW Droege); (PCYU); 3 ♂, Tompkins Co., Buttermilk Falls S.P., Ithaca, 7.x.1967, (G & K Eickwort); 1 ♀, Tompkins Co., 6-mile Creek, SE Ithaca Reservoir, 25.v.1968, (G & K Eickwort); 1 ♂, Tompkins Co., Michigan Hollow, gravel pit, 5mi S of Danby, 7.ix.1968, (G & K Eickwort); 1 ♀, Albany Co., Partridge Run St. Game Area, 5mi N Rensselaerville, 6.vi.1970, (G & K Eickwort); 1 ♀, Albany Co., Rensselaerville, 28.vii.1970, (G & K Eickwort); 1 ♀, Albany Co., Rensselaerville, Huyck Reserve, 12.vi.1969, (G & K Eickwort); 1 ♂, Albany Co., Colonie, 20.viii.1969, (G & K Eickwort); 1 ♀, Cayuga Co., Fair Haven Beach S.P., 8.vi.1968, (G & K Eickwort); 1 ♀, Ludlowville, 6.vi.1968, (LL Pechuman); 1 ♂ & 3 ♀, Nassau Co., Jones Beach S.P., 31.vii.1974, (GC Eickwort); 1 ♂ & 1 ♀, Nassau Co., Jones Beach S.P., 26.vi.1976, (G Eickwort); 7 ♂, Nassau Co., Hempstead Lake S.P., 4–6.vii.1974, (GC Eickwort); 2 ♀, Nassau Co., Kennedy Wildlife Sanct., Tobay Beach, 18.vi.1989, (GC Eickwort); 1 ♀, Nassau Co., Tobay Beach, 24–26.vi.1976, (GC Eickwort); 1 ♂, Nassau Co., Floral Pk., 4.vii.1982, (D Yanega); 1 ♀, Montauk, 4.v.1947, (R Latham); 1 ♀, 3-mile Har., 6.vi.1941, (R Latham); 1 ♂, Van Cortland Pk., 20.vii.1913 (CUIC); 1 ♂, Queens Co., Floral Pk., Long I., 6.viii.1983, (D Yanega) (SEMC); NEW JERSEY: 1 ♀, Atlantic Co., 39 35’N 74 46’W, 21.vii.2003, (B Ahlstrom) (PCYU); 1 ♂, Weymouth, 26.vii.1923; 1 ♀, San Isle Junction, “May,25” (CUIC); 1 ♂, Ramsey, 10.vii.1913 (SEMC); MARYLAND: Camp Springs, 11.v.10, (JC Crawford); 1 ♀, Cabin John, 16.iv.1915, (JC Crawford); 1 ♀, Pr. George’s Co., N38.9764 W76.7491 20.vii.2002, (S Kolski); 1 ♀, Pr. George’s Co., N38.9591 W76.734 20.viii.2004, (S Kolski); 1 ♀, Pr. George’s Co., N39.002 W76.7505 20.viii.2004, (S Kolski); 3 ♀, Pr. George’s Co., N38.9977 W76.7573 30.ix–1x,2004, (S Kolski); 4 ♀, Anne Arundel Co., N38.7839 W76.7014, B Hollister (SEMC); 1 ♀, Calvert Co., N38.536 W76.518, 7.vii.2006, (M Gates); 1 ♀, Talbot Co., N38.8 W76.283, 7–8.v.2005, (W Steiner); 1 ♀, Caroline Co., N38.9194 W75.8279, 13.v.2007, (M Price); 1 ♂, Wicomico Co., N38.2909 W75.5364, 12.vi.2004, (SW Droege); 1 ♂, Anne Arundel Co., N38.7839 W76.7014, 21.ix.2004, (B Hollister); 1 ♂, Pr. George’s Co., N38.9893 W76.7322, 20.vii.2004, (S Kolski); 1 ♂ & 1 ♀, Pr. George’s Co., N38.9123 W76.755, 12–13.viii.2003, (Haramis & Archer); 2 ♀, Pr. George’s Co., N38.9123 W76.755, 13–14.viii.2003, (Haramis & Archer); 1 ♀, Pr. George’s Co., N38.9123 W76.755, 28–29.viii.2003, (Haramis & Archer); 1 ♀, Pr. George’s Co., N38.9123 W76.755, 26–27.viii.2003, (Haramis & Archer); 2 ♀, Pr. George’s Co., N38.9123 W76.755, 14–15.viii.2003, (Haramis 16 · Zootaxa 2032 © 2009 Magnolia Press GIBBS FIGURE 7. Lasioglossum tegulare terminal structures of male. Diagram of genitalia with ventral and dorsal sides pictured on left and right, respectively (above), diagram of S7 (middle) and diagram of S8 (below). LASIOGLOSSUM (DIALICTUS) TEGULARE SPECIES GROUP Zootaxa 2032 © 2009 Magnolia Press · 17 & Archer); 1 ♀, Pr. George’s Co., N38.9959 W76.7886, 2.ix.2004, S Na; 1 ♀, Caroline Co., N39.1098 W75.7724, 7.iv.2005, (SW Droege) (PCYU); 6 ♀, Prince Georges Co., Temple Hills, 22.viii.1976, (GC Eickwort) (CUIC); WASHINGTON, D.C.: 2 ♀, N38.931 W77.116, 24.vi.2006, Pascarella; 1 ♂, N77.034 W38.885, 18–19.x.2004, (E Keto); 1 ♂, N38.9463 W77.0344, 16.vi.2004, (SW Droege); 1 ♀, N38.8871 W77.0128, 1.ix.2005, (NB Staff); 1 ♀, Mall, N38.8912 W777.0242, 16.viii.2006, (SW Droege); 1 ♂, N38.891 W77.0308, 15–16.vii.2004, (C Osborn); 1 ♂, N38.879 W77.0333, 13–14.vii.2004, (C Osborn); 1 ♂, N77.0261 W38.8694, 4–5.x.2004, (E Keto) (PCYU); VIRGINIA: 3 ♀, Assateague I., N37.9625 W75.3108, 30.vi–1.vii.2006, (SW Droege) [Barcoded]; 1 ♀, Assateague I., N37.9576 W75.3147, 30.vi–1.vii.2006, (SW Droege); 1 ♀, Assateague I., N37.9377 W75.3177, 30.vi–1.vii.2006, (SW Droege) [Barcoded]; 1 ♀, Assateague I., N37.9486 W75.3136, 2.vii.2006, (SW Droege); 1 ♀, Assateague I., N37.9086 W75.3564, 1–2.vii.2006, (SW Droege); 1 ♀, Virginia Beach, N36.917 W75.3564, 16–17.vi.2007, (W Steiner); 1 ♀, Fluvanna Co., N37.753 W78.162, 2.x.2004, (SW Droege) [Barcoded]; 1 ♀, Accomack Co., N37.938 W75.318, 30.vi–1.vii.2006, (SW Droege); 1 ♀, Hwy 340, 10km N of Shenandoah, N38.564 W78.606, 7.vi.2005, (A Zayed) [Barcoded] (PCYU); 2 ♀, Clarke Co., Blandy Exp. Farm E Boyce, 12–14.vi.1986, (JK Liebherr) (CUIC); WEST VIRGINIA: 2 ♀, Hampshire Co., N39.346 W78.403, 29–30.v.2004, (SW Droege) [Barcoded]; 1 ♀, Hampshire Co., N39.351 W78.509, 29–30.v.2004, (SW Droege) [Barcoded]; 1 ♂, Hampshire Co., N39.2334 W78.6843, 11.vii.2002, (SW Droege); 1 ♂, Hampshire Co., N39.31 W78.54, 11.vii.2002, (SW Droege); 1 ♂, Hampshire Co., N39.3012 W78.4358, 11.vii.2002, (SW Droege); 1 ♂, Hampshire Co., N39.4401 W78.4872, 7.vii.2002, (SW Droege); 1 ♂, Hampshire Co., N39.2886 W78.4819, 11.vii.2002, (SW Droege) (PCYU); 1 ♀, Hampshire Co., N39.415 W78.5012, 7.vii.2002, (SW Droege); 1 ♀, Hampshire Co., N39.333 W78.4585, 6.vii.2002, (SW Droege) (SEMC); KENTUCKY: 15 ♀, Wayne Co., N39.924 W84.8715, 23–24.vii.2007, (SW Droege); 1 ♀, Laurel Co., N37.1528 W84.1167, 27.vii.2007, (SW Droege) (PCYU); TENNESSEE: 1 ♀, Rutherford Co., N35.8275 W86.2912, 20.vii.2007, (D Green); 1 ♀, Rutherford Co., N35.8197 W86.3159, 20.vii.2007, (D Green) (PCYU); NORTH CAROLINA: 1 ♀, Union Co., N34.984 W80.449, ix–x.2003, (R Jackowski) [Barcoded]; 1 ♀, Great Smoky Mountains National Park, Cataloochee overlook, N35.54 W83.06, 6.viii.2006, (J Gibbs); 1 ♂, Highlands, 22.vii.1958, (TB Mitchell); 1 ♀, Pettigrew S.P., 27.v.1959, (TB Mitchell) (PCYU); 6 ♂ & 1 ♀, Beutenmuller, Black Mts., viii.1912; SOUTH CAROLINA: 1 ♂ & 1 ♀, Okanee Co., near Walhalla, N34.813 W83.137, 9.viii.2006, (J Gibbs) [Barcoded]; 1 ♀, Chesterfield Co., N34.637 N80.176, 18–19.v.2006, (SW Droege); 5 ♀, Chesterfield Co., N34.55 W80.26, 2007, (L Housh) (PCYU); GEORGIA: 2 ♀, Athens, 15.vi.1909, (JC Bradley); 1 ♂, Cobb Co., Lost Mount, 13.vii.1913; 1 ♂, Rabun Bald, 4000–4800ft, 21.viii.1913; CANADA, ONTARIO, 1 ♂, Norfolk, N42.6497 W80.5729, 11.viii.2007, (A Taylor); 5 ♀, Norfolk, N42.6493 W80.5687, 11.vi.2007; 1 ♂, Caledon, Gschwendtner property, N43.8148 W79.9768, 18.ix.2003, (J Grixti) (PCYU). Type depository. ANSP. Etymology. No explanation is given for the name in the original description but the species was undoubtedly named for the remarkable shape of the tegula. Comments. The range of L. tegulare is evidently more restricted than previously reported (Moure & Hurd 1987). Northwestern and southeastern records likely correspond to one or other of the species described below. Some records from Texas could be misidentifications of L. coactum (Cresson), which differs in having deeply impressed marginal zones of T1 and T2 in both sexes. Lasioglossum (Dialictus) ellisiae (Sandhouse), comb. n. (Figures 8A–D) Halictus (Chloralictus) ellisiae Sandhouse, 1924: 11. ♀. Lasioglossum (Chloralictus) ellisiae: Michener, 1951: 1113 (catalogue). Dialictus tegularis Mitchell, 1960: 423 (synonymy). 18 · Zootaxa 2032 © 2009 Magnolia Press GIBBS Diagnosis. Females of L. ellisiae are unique in having the integument of the mesoscutum (especially adjacent to the parapsidal lines) and mesepisternum smoother and more reflective than those of other species of the tegulare group in eastern North America. The remaining four species each have the integument in these areas roughened and dulled due to microsculpture. Females of L. ellisiae can be further distinguished by the following characters: head and mesosoma primarily bluish-green, paraocular area with sparse subappressed hairs, and inner hind tibial spur with 3 or 4 teeth (not including apex of rachis). Females of L. puteulanum are deep blue in colour. Females of L. lepidii have dense subappressed hairs on the paraocular area which obscure the surface adjacent to the inner eye margin and lower paraocular area. Females of L. carlinvillense have only 2 teeth on the inner hind tibial spur. Males of L. ellisiae can be distinguished from the other species by the sparsely punctate zones of T2 and T3 anterior of the premarginal line. The males of L. tegulare, L. lepidii, and L. puteulanum all have uniformly dense punctation basal to the premarginal lines of T2 and T3. Redescription. Female. Length: 5.25 (4.6–5.5) mm, fore wing length: 3.5 (3.1–3.7) mm, head length: 1.3 (1.1–1.4) mm, head width: 1.4 (1.2–1.4) mm, n=9 Colouration. Head and mesosoma dull metallic bluish-green except the following: mandible base brown- piceous, apex red; clypeus brown-piceous below, golden above; supraclypeal area green below, gold above; antenna brown-piceous, ventral surface brown, F8–F10 ruddy brown to testaceous; mesoscutum green with gold reflections; tegula brown-piceous, central area ferruginous; legs brown-piceous, fore, medio- and distitarsi reddish, mid and hind distitarsi ruddy brown; wing venation and pterostigma testaceous-brown; wings very faintly dusky; propodeum bluer than mesoscutum; metasoma brown-piceous. Pubescence. Head and mesosoma with sparse, erect, plumose hairs (1–1.5OD), longer on metanotum and ventral pleura (2OD); posterolateral margin of pronotum and pronotal lobe with dense, appressed tomentum; dense scopa on hind femur; lateral surface of propodeum with long branched hairs (2OD); acarinarial appressed fan complete; terga with sparse, erect hairs (1–2OD), more abundant on ventrolaterally reflexed areas; T3–T5 ventrolaterally reflexed areas with few, erect hairs (2.5–3OD); T2–T3 basolateral portions and T4 dorsal surface with sparse appressed, plumose hairs; sterna with long, posteriorly oriented hairs emerging from apical half of disc (2–3OD); S1–T4 hairs with long branches. Surface sculpture. Clypeus glabrate except upper margin imbricate, punctures moderately coarse below (i=1–1.5d), fine above (i=d); supraclypeal area smooth and shining, margins imbricate, punctures fine, irregularly spaced (i=1–4d); lower paraocular area imbricate, smooth and shining below, punctures moderately coarse and deep (i LASIOGLOSSUM (DIALICTUS) TEGULARE SPECIES GROUP Zootaxa 2032 © 2009 Magnolia Press · 19 FIGURE 8. Lasioglossum ellisiae A) face of female holotype; B) lateral habitus of female holotype; C) face of male; D) lateral habitus of male. Male. Length: 4.0–4.3 mm, fore wing length: 3.1 mm, head length: 1.2 mm, head width: 1.2–1.3 mm, n=2 Colouration. Head and mesosoma dark metallic blue except lower paraocular area and medial areas of mesoscutum and mesoscutellum with golden-green reflections; the following dark brown-piceous: labrum; mandible except apex red; lower clypeus; antenna; tegula except central area ferruginous; legs except medio- and distitarsi brown; metasoma; wing venation and pterostigma brown; wings hyaline except apex faintly dusky. Pubescence. Paraocular area and interantennal area with sparse, subappressed hairs; sparse longer erect hairs on head, mesosoma, anteriorly directed surface of T1 and ventrolaterally reflexed portions of terga (1.5–2OD), longest on metanotum; T1–T4 with short laterally oriented setae; sterna with erect hairs, densest on S4–S5. Surface sculpture. Head and mesosoma smooth and shining; clypeus (i 20 · Zootaxa 2032 © 2009 Magnolia Press GIBBS (i LASIOGLOSSUM (DIALICTUS) TEGULARE SPECIES GROUP Zootaxa 2032 © 2009 Magnolia Press · 21 Whiteside Co., Morrison, 10.vii.1968, (JC Marlin); 1 ♀, Mason Co., Bath, 2.vii.1968, (JC Marlin); 1 ♀, Mason State Forest, 2.vi.1966, (WJ Knee); 1 ♀, McHenry Co., Chain O’ Lakes St. Pk., 22.vi.1967, (Laberge & Ribble); 1 ♀, Lake Co., 2 mi NW Volo, 22.vi.1967, (Laberge & Ribble); 1 ♀, Long L., “bog, smtwd.” 11.viii.1906; 10 ♀, Algonquin, 16.vi.1909, (Nason); 2 ♀, Algonquin, 24.vi.1909, (Nason); 1 ♀, Woodford Co., 9mi N East Peoria, 7.viii.1968, (JC Marlin); 1 ♂, Macoupin Co., Plainview, 22.vii.1915 (INHS); 1 ♀, Kankakee Co., Hooper Br, 2.vii.2003, (RP Jean) (PCYU); WISCONSIN: 1 ♀, Sauk Co., Spring Green Preserve, N43.19785 W90.05904, 23.vii.2006, (A Wolf) (UWGB); MINNESOTA: 2 ♀, Clay Co., 3mi E & 2mi S of Felton, N47.047 W96.438, blue and yellow pan traps, 21.vii.2006, (RL Andres) (PCYU); CANADA, ONTARIO: 1 ♀, Norfolk Co., Pterophylla Plant Nursery, N42.384 W80.344, malaise trap, 13–22.viii.2006, (PJ Carson) [Barcoded]; 2 ♀, Toronto, York University, N43.775 W79.504, 24.v.2006, (J Gibbs) [Barcoded]; 1 ♀, Toronto, Ulster St. parquette, N43.659 W79.413, x.2006, (J Gibbs); 1 ♀, Haldimane-Norfolk Co., Nixon W Pr, 11.v.1998, H Douglas; 1 ♀, Haldimane-Norfolk Co., Nixon W Pr, 28.v.1998, (H Douglas) (PCYU). Etymology. This species was named for Marion Durbin Ellis who described a number of Dialictus and other bee species. Type depository. NMNH Cat. No. 26400. Comments. Specimens that could be attributed to this species extend its range into the great plains of the United States. Additional western species that could be mistaken for L. ellisiae may co-occur in this area. Further study will be needed to test species boundaries of the western fauna and to determine the western range limits of L. ellisiae. Lasioglossum (Dialictus) lepidii (Graenicher), comb. n. (Figures 9A–D) Halictus (Chloralictus) lepidii Graenicher, 1927: 204. ♀ ♂. Lasioglossum (Chloralictus) lepidii: Michener, 1951: 1114 (catalogue). Dialictus tegularis Mitchell, 1960: 423 (synonymy). Diagnosis. Females of L. lepidii can be distinguished by the following combination of characters: head and mesosoma pale to golden green, paraocular area partially obscured by appressed hairs along inner eye margin, distinct microsculpture between punctures of mesoscutum and mesepisternum, and three teeth on the inner hind tibial spur (not including apex of rachis). Females of L. puteulanum have the head and mesosoma deep blue. Females of L. tegulare have sparse subappressed hairs on the paraocular area which do not obscure the surface. Females of L. ellisiae have the integument of the mesoscutum (particularly adjacent to parapsidal lines) and mesepisternum smooth, with at most faint microsculpture which gives these areas a shiny appearance. Females of L. carlinvillense have only two teeth on the inner hind tibial spur. Males of L. lepidii are unique among these species in having dense, appressed hairs that obscure large areas of the face including the paraocular area, clypeus and frons. Males of other species have more limited tomentum on the face often only obscuring the paraocular area and never obscuring the frons. Redescription. Female. Length: 4.3 (4.3–4.9) mm, fore wing length: 3 (3.0–3.2) mm, head length: 1.3 mm, head width: 1.3 (1.3–1.4) mm, n=2 Colouration. Head and metasoma dull metallic blue-green; mandible base brown, apex red: clypeus brown below, golden-green above; supraclypeal area golden-green; lower paraocular area brown-piceous below; antennae brown, apical flagellomeres orange-yellow ventrally; mesoscutum green with hints of gold; tegula brown-piceous, central area ferruginous; legs brown-piceous, fore medio- and distitarsi testaceous, mid and hind medio- and distitarsi ruddy brown; wing venation and pterostigma testaceous-brown; wings faintly dusky; dorsal surface of propodeum blue; metasoma brown-piceous. Pubescence. Head and mesosoma with sparse, erect, plumose hairs (1–1.5OD), longer on metanotum and ventral pleura (2OD); mid to lower paraocular area with appressed tomentum; posterolateral margin of 22 · Zootaxa 2032 © 2009 Magnolia Press GIBBS pronotum and pronotal lobe with dense, appressed tomentum; dense scopa on hind femur; lateral surface of propodeum with long branched hairs (2OD); acarinarial appressed fan complete; terga with sparse erect hairs (1–2OD), more abundant on ventrolaterally reflexed portions; T3–T5 ventrolaterally reflexed areas with hairs longer (2.5–3OD); T2–T3 basolateral portions and T4 dorsal surface with sparse appressed, plumose hairs; sterna with long, posteriorly oriented hairs emerging from apical half of disc (2–3OD); S1–S4 hairs with long branches. Surface sculpture. Clypeus glabrate below, upper margin imbricate, punctures moderately coarse below (i=1–2d), fine above (i=d);; supraclypeal area smooth, margins imbricate, punctures fine, irregularly spaced (i=1–1.5d); lower paraocular area imbricate, glabrate below, punctures moderately fine (i LASIOGLOSSUM (DIALICTUS) TEGULARE SPECIES GROUP Zootaxa 2032 © 2009 Magnolia Press · 23 onto lateral slope; lateral surface of propodeum scabriculous with obscure but moderately fine and deep and close punctures (i=1–1.5d); posterior surface of propodeum smoother with distinct punctures (i=1–2d); metasoma faintly coriarious; terga with fine but distinct punctures (i=1–1.5d), apical impressed areas impunctate; anteriorly directed surface of T1 largely impunctate. FIGURE 9. Lasioglossum lepidii A) face of female; B) lateral habitus of female holotype; C) face of male; D) lateral habitus of male. Structure. Face as long as broad; eyes convergent below (UOD:LOD = 6.5:4.0); carina of frontal line ending less than 2OD from median ocellus; OOC distinctly less than IOC; eye wider than gena from lateral view; hypostomal carinae parallel; pedicel subequal in length to F1; F2–F10 length 1.5 times breadth, F1 very slightly longer than F2; ratio of lengths of mesoscutellum: metanotum: dorsal surface of propodeum (2.1:1.2:1.5); tegula enlarged, posterior margin not strongly angled; propodeal carina weak on lateral portions of margin between dorsal and posterior surfaces; metasoma narrow relative to female. Terminalia. As in L. tegulare see Fig. 7A–C Range. Florida. Specimens examined. USA, FLORIDA, HOLOTYPE ♀, South Miami, 20.iv.1927 (NMNH); 1 ♂, Miami, 4.v.1927, (S Graenicher); 1 ♀, Miami, 22.vi.192?, (S Graenicher); Highlands Co., Highlands Hammock St. Pk., 13.iv.1964, (GC Eickwort); 1 ♀, Islamorada, 12.iv.1966, G Eickwort; (SEMC); 1 ♀ and 2 ♂, Westchester, Miami, 31.viii.2005, (JA Genaro) [Barcoded]; 1 ♀, Westchester, Miami, 31.viii.2005, (JA 24 · Zootaxa 2032 © 2009 Magnolia Press GIBBS Genaro) (PCYU); 3 ♀, Wakulla Co., 2mi N of Mack’s Landing, Aalachicola Nat. For. 21.v.1981, (GC Eickwort et al.); 2 ♀, Wakulla Co., Ochlockones River S.P. 21.v.1981, (GC Eickwort et al.); 1 ♂ & 2 ♀, Collier Co., Seminole S.P. 25–26.v.1978, (NF & JB Johnson); 1 ♀, Archbold Biol. Stat., Lk. Placid, Highlands Co., 2.iv.1984, (B Alexander); 3 ♂ & 2 ♀, Monroe Co., Key Largo (city), 22.iii.1987, (Eickwort & Spielholz); 4 ♂ & 3 ♀, Monroe Co., Bahia Honda Key, Bahia Honda St. Rec. Area, 25.iii.1987, (Eickwort & Spielholz); 1 ♂ & 3 ♀, Monroe Co., Key Largo, Pennekamp S.P., 22.iii.1987, (Eickwort & Spielholz); 1 ♂, Monroe Co., Key Largo (east end), 22.iii.1987, (Eickwort & Spielholz); 2 ♂ & 4 ♀, Monroe Co., Long Key, Long Key St. rec. Area, 23.iii.1987, (Eickwort & Spielholz); 1 ♂, Dade Co., Redlands, 21.iii.1987, (Eickwort & Spielholz); 2 ♀, Broward Co., Hallandale Beach, 10.xii.1985, (GC Eickwort); 1 ♀, Leon Co., Tall Timbers Res. Stat., 3mi E Iamonia, 30.iii.1986, (B Alexander); 1 ♀, Pinellas Co., Ft. Desoto. Co. Pk, 1.vi.1978, (NF & JB Johnson); 1 ♂, Wakulla Co., Sopchoppy, 1.iv.1981, (LL Pechuman) (CUIC). Etymology. No explanation for the name is given in the original description but likely refers to the flowers of Lepidium virginicum L. that (among others) the original specimens were collected from. Type depository. NMNH Cat. No. 41800. Comments. In some cases, species from Caribbean islands are known to also occur in Florida. Dr. Julio Genaro, the expert on Caribbean Apoidea, is unaware of any species matching the description of this species in the neighbouring islands (J.A. Genaro; personal communication). Lasioglossum (Dialictus) puteulanum Gibbs, sp. n. (Figures 10A–D) Diagnosis. Females of L. puteulanum are unique among these species in having the head and mesosoma distinctly blue, whereas other species are typically pale or golden green to at most faintly bluish-green. The head is on average slightly longer (often longer than wide) (ratio = 0.95: 1.10) and the clypeus protrudes giving the face a more triangular appearance than other species. In contrast, females of other species often have the head wider than long (ratio = 0.85: 1.0) with a much rounder appearance of the face. The males of L. puteulanum can be recognized by the following combination of characters: head and mesosoma blue, ventral surface of flagellum pale, appressed hairs of face mostly limited to paraocular areas and only partially obscuring clypeus, and T2–T3 punctures uniformly dense on disc basal to the premarginal line. Males of L. ellisiae have the ventral surface of the flagellum dark to ferruginous and T2–T3 punctures dense on basal half but sparse approaching premarginal line. Males of L. lepidii have the ventral surface of the flagellum bright yellow and appressed hairs of the face dense, obscuring the majority of the clypeus and frons. The subappressed hairs of the face of L. tegulare are usually limited to the lower paraocular area but are more evenly distributed across the face in L. puteulanum. Description. Female. Length: 5.2 (4.3–5.5) mm, fore wing length: 3.6 (2.9–3.6) mm, head length: 1.4 (1.2–1.4) mm, head width: 1.4 (1.2–1.4) mm, n=10 Colouration. Head and mesosoma dull metallic blue except the following: mandible base brown, apex red: clypeus brown brown-piceous below; lower paraocular area below piceous-brown; antennae brown- piceous, flagellum ventral surface paler, F8–F10 orange-yellow ventrally; mesoscutum pale blue; tegula piceous, central area ferruginous; legs brown-piceous, medio- and distitarsi ruddy brown; wing venation and pterostigma testaceous-brown; wings very faintly dusky; metasoma brown-piceous. Pubescence. Head and mesosoma with sparse, erect, plumose hairs (1–1.5OD), longer on metanotum and ventral pleura (2OD); mid to lower paraocular area with appressed tomentum; posterolateral margin of pronotum and pronotal lobe with dense, appressed tomentum; dense scopa on hind femur; lateral surface of propodeum with long branched hairs (2OD); acarinarial appressed fan complete; terga with sparse, erect hairs (1–2OD), more abundant on ventrolaterally reflexed portions; T3–T5 ventrolaterally reflexed areas with few erect hairs (2.5–3OD); T2–T3 basolateral portions and T4 dorsal surface with sparse appressed, plumose hairs; sterna with long, posteriorly oriented hairs emerging from apical half of disc (2–3OD); S1–S4 hairs LASIOGLOSSUM (DIALICTUS) TEGULARE SPECIES GROUP Zootaxa 2032 © 2009 Magnolia Press · 25 with long branches. Surface sculpture. Clypeus glabrate below, upper margin imbricate, punctures moderately coarse below (i=1–2d), fine above (i=d); supraclypeal area imbricate, punctures fine, well spaced but not sparse (i=1–2d); lower paraocular imbricate, glabrate below, area punctures moderately coarse and deep (i 26 · Zootaxa 2032 © 2009 Magnolia Press GIBBS evident between dorsal and posterior surfaces; metasoma narrow relative to female. Terminalia. As in L. tegulare see Fig. 7A–C. FIGURE 10. Lasioglossum puteulanum A) face of female holotype; B) lateral habitus of female holotype; C) face of male; D) lateral habitus of male. Range. Coastal plain of North Carolina to Florida Specimens examined. USA, FLORIDA, HOLOTYPE ♀, Palm Beach Co., N26.34889 W80.2756, 25.i.2005, (SW Droege) [Barcoded]; 1 ♀, collected with holotype [Barcoded]; 1 ♀, Miami, Westchester, 31.viii.2006, (JA Genaro) [Barcoded]; 1 ♀, Hendry Co., N26.3121 W81.2354, 27.i.2005, (SW Droege) [Barcoded]; 1 ♂, Broward Co., N26.30345 W80.20219, 25.i.2005, S.W. Droege [Barcoded]; 3 ♀ & 2 ♂, Broward Co., N26.107 W80.264, 27.i.2005, (SW Droege) [Barcoded]; 13 ♀, Martin Co., N27.083 W80.1442, 4.vi.2007, (SW Droege); 8 ♀, Martin Co., N27.1008 W80.152, 4.vi.2007, (SW Droege); 2 ♂, Martin Co., N27.081 W80.1416, 4.vi.2007, (SW Droege); (PCYU); 1 ♀, Inverness, (C Robertson) (INHS); 3 ♂, Gainesville, 6.v.1955, (HE & MA Evans); 1 ♂, Brighton, 19.iv.1937, (JC Bradley); 1 ♂, Crescent City, 1–3.v.1955, (HE & MA Evans); 4 ♂, Wakulla Co., 2mi N of Mack’s Landing, Apalachicola Nat. For. 21.v.1981, (GC Eickwort et al.); 1 ♀, Wakulla Co., Ochlockones River S.P. 21.v.1981, (GC Eickwort et al.); 1 ♂ & 1 ♀, Collier Co., Seminole S.P. 25–26.v.1978, NF&JB Johnson; 1 ♀, Archbold Biol. Stat., Lk. Placid, Highlands Co., 2.iv.1984, (B Alexander); 3 ♂, Monroe Co., Key Largo (city), 22.iii.1987, (Eickwort & Spielholz); 1 ♀, Monroe Co., Key Largo, Pennekamp S.P., 22.iii.1987, (Eickwort & Spielholz); 1 ♂, Monroe LASIOGLOSSUM (DIALICTUS) TEGULARE SPECIES GROUP Zootaxa 2032 © 2009 Magnolia Press · 27 Co., Big Pine Key, Key Deer NW Ref., 25.iii.1987, (Eickwort & Spielholz); 1 ♂ & 4 ♀, Broward Co., Broward Beach S.P., Fort Lauderdale, 9.xii.1985, (GC Eickwort); 15 ♀, Broward Co., Hallandale Beach, 10.xii.1985, (GC Eickwort); 1 ♀, Leon Co., Tall Timbers Res. Stat. 3mi W Iamonia, 30.iii.1986, (B Alexander); 1 ♀, Dade Co., Matherson Hammock Co. Pk. 20–21.iii.1987, (Eickwort & Spielholz) (CUIC); 3 ♂ & 1 ♀, Highlands Co., Hghlds., Hammock S.P., 14.iv.1964, (GC Eickwort); 1 ♀, Liberty Co., Torreya S.P., 6.iv.1964, (GC Eickwort); 2 ♀, Franklin Co., coast 10mi S of Panacea, 7.iv.1964, (GC Eickwort); 7 ♀, Broward Co., Hollywood, 13.xii.1985, (CD Michener); 1 ♂, Collins, Seminole S.P., 13.iv.1986, (GC Eickwort) (SEMC); GEORGIA, 1 ♂, Bainbridge, vii.1909; 2 ♀, Coquitt Co., Reed Bingham S.P., 22.v.1981, (GC Eickwort et al.); 1 ♀, Colquitt Co., Reed Bingham S.P., 23.v.1981, (GC Eickwort et al.); 1 ♀, Colquitt Co., Murphy, 5.iv.1981, (LL Pechuman) (CUIC); SOUTH CAROLINA, 1 ♀, C. Sandhills NWR, N34.547 W80.177, 6–7.ix.2005, (SW Droege) [Barcoded]; 1 ♂, C. Sandhills NWR, N34.56 W80.256, 6.ix.2006, (SW Droege); 2 ♀, Chesterfield Co., N34.623 W80.19, 18–19.v.2006, (SW Droege); 1 ♀, Chesterfield Co., N34.637 W80.176, (SW Droege) [Barcoded] (PCYU); NORTH CAROLINA, 1 ♀, Moore Co., N35.284 W79.314, 19.v.2006, (SW Droege) [Barcoded] (PCYU); 1 ♂, Raleigh, 16.vii.1948, (MW Wing); 1 ♂, Wake Co., 10.vii.1949, (MW Wing) (CUIC); 1 ♂, TENNESSEE, 4.vi.1918 (CUIC). Type depository. PCYU Etymology. The specific epithet refers to the blue colouration of the head and mesosoma. Comments. This species may be extremely difficult to differentiate from L. tegulare in areas where their ranges overlap. The blue colouration characteristic of this species should be sufficient to identify most individuals; however, colouration is sometimes variable in Dialictus and can be affected by preservation methods. Species identification of questionable specimens is possible using DNA barcodes. Lasioglossum (Dialictus) carlinvillense Gibbs, sp. n. (Figures 11A–D) Diagnosis. This species is smaller in body size than other species. The inner hind tibial spur of the female is unique in having only two subapical teeth as opposed to the three or four teeth in the other species. The male is unknown. Description. Female. Length: 4.3 (4.3–4.5) mm, fore wing length: 2.7 (2.7) mm, head width: 1.2 (1.2–1.3) mm, head length: 1.2 (1.2) mm, n=3 Colouration. Head and mesosoma dull metallic bluish-green except the following: labrum brown-piceous, mandible base brown, apex red; clypeus brown, golden above; supraclypeal area bronzed above; antennae brown-piceous except ventral surface of flagellomeres brown, F8–F10 testaceous ventrally; gena blue; mesoscutum green to golden green; tegula piceous with central area ferruginous; legs brown-piceous, medio- and distitarsi ferruginous; wing venation and pterostigma testaceous; wings faintly dusky; propodeum darker with blue reflections; metasoma piceous-brown; apical portions of terga and sterna light brown. Pubescence. Lower paraocular area with sparse, subappressed, plumose hairs; head and mesosoma with sparse, erect, plumose hairs (1–1.5OD), longer on metanotum and ventral pleura (2OD); posterolateral margin of pronotum and pronotal lobe with dense, appressed tomentum; dense scopa on hind femur; propodeal lateral surface hairs (2OD) with long branches; acarinarial appressed fan complete; terga with sparse, erect hairs (1–2OD), more abundant on ventrolaterally reflexed portions; T3–T5 ventrolaterally reflexed areas with few erect hairs (2.5–3OD); T2–T3 basolateral portions and T4 dorsal surface with sparse appressed, plumose hairs; sterna with long, posteriorly oriented hairs emerging from apical half of disc (2–3OD); S1–S4 hairs with long branches. Surface sculpture. Clypeus glabrate except upper margin imbricate, punctures moderately coarse (i=1–2d), fine above (i=d); supraclypeal area smooth and shining below, imbricate above, punctures fine (i=1–1.5d); lower paraocular area imbricate, glabrate below, punctures moderately coarse and deep (i 28 · Zootaxa 2032 © 2009 Magnolia Press GIBBS and obscure; mesoscutum and mesoscutellum tessellate between fine punctures; mesoscutum punctures well spaced but not sparse in anteromedial and submedial areas (i=1–1.5d), dense on remainder of disc (i FIGURE 11. Lasioglossum carlinvillense A) face of female holotype; B) lateral habitus of female holotype. Range. South-central Illinois Specimens examined. USA, ILLINOIS: HOLOTYPE ♀, Macoupin Co., E of Carlinville, N39.2787 W89.7961, 25.vi.2006, (J Gibbs & C Sheffield) [Barcoded]; 1 ♀, Carlinville, N39.2787, W89.8898, 24.vi.2006, (J Gibbs) [Barcoded]; 1 ♀, Litchfield, N39.1484 W89.667, 25.vi.2006, (C Sheffield) [Barcoded] (PCYU); 1 ♀, Dubois, 8.viii.1917 (INHS) Type depository. PCYU Etymology. The specific epithet refers to the type locality and the famous collection site of Charles Robertson. Comments. The male of this species is unknown. The known range of this species is very small. It is possible that this is a prairie species found at the eastern edge of its range. LASIOGLOSSUM (DIALICTUS) TEGULARE SPECIES GROUP Zootaxa 2032 © 2009 Magnolia Press · 29 FIGURE 12. Mesepisternum of female showing microsculpture of (A) L. tegulare and (B) L. ellisiae. 30 · Zootaxa 2032 © 2009 Magnolia Press GIBBS FIGURE 13. Dorsal surface of the propodeum showing surface sculpture of the metapostnotum in (A) L. lepidii and (B) L. tegulare. LASIOGLOSSUM (DIALICTUS) TEGULARE SPECIES GROUP Zootaxa 2032 © 2009 Magnolia Press · 31 Key to eastern species The following keys to species can be substituted for L. tegulare in couplet 6 for females and couplet 4 for males in Mitchell’s (1960) key to Dialictus. Key to females. 1 Integument of head and mesosoma with distinct metallic blue reflections; head slightly longer than broad to sub- equal (ratio = 0.95: 1.10) (Fig. 10A); clypeus protruding two thirds below suborbital tangent (Southeastern USA) ...... L. puteulanum - Integument of head and mesosoma with metallic green reflections, if blue reflections present then usually restricted to pleura and propodeum; head usually broader than long (ratio = 0.85: 1.0) (e.g. Fig. 6A); clypeus one half below suborbital tangent ...... 2 2 Surface of mesepisternum (Fig. 12A) and mesoscutum adjacent to parapsidal lines smooth and shining, only faint microsculpture between punctures (Northeastern USA and Southern Ontario)...... L. ellisiae - Surface of mesepisternum (Fig. 12B) and mesoscutum dull and roughened due to microsculpture between punctures ...... 3 3 Inner hind tibial spur with only two subapical teeth (Illinois)...... L. carlinvillense - Inner hind tibial spur with three or four subapical teeth...... 4 4 Paraocular area with appressed hair dense, obscuring surface along inner margin of eye (Fig. 9A); metapostnotum with anastomozing rugae, medial striation not distinct (Fig. 13A) (Florida) ...... L. lepidii - Paraocular area with appressed hairs sparse not obscuring surface (Fig. 6A); metapostnotum striate (Fig. 13B), if submedial striations anastomozing then long and straight medial striation distinctly visible (Northeastern USA and Southern Ontario)...... L. tegulare Key to males. 1 Ventral surface of antenna dark brown to ferruginous; punctures on T2–T3 dense on basal portion of disc becoming extremely sparse and indistinct towards the impunctate apically impressed area (Fig. 14A) (Northeastern USA and Southern Ontario)...... L. ellisiae - Ventral surface of antenna somewhat dull to bright yellow; punctures on T2–T3 uniformly dense from margin to impunctate apically impressed region (Fig. 14B) ...... 2 2 Integument of face (including paraocular area, frons and majority of clypeus) obscured by white tomentum (Fig. 9C); flagellomeres bright yellow on ventral surface; antennal sockets nearer to inner eye margin than each other (ratio = 0.7); pre-episternum punctate-reticulate (Florida) ...... L. lepidii - Integument of face less obscured by tomentum primarily on paraocular area (never extending onto frons) (Figs. 6C, 10C); flagellomeres yellowish-brown to yellow ventrally; distance between antennal sockets and inner margin of eye subequal to distance between eye sockets (ratio = 0.9–1.0); pre-episternum rugulose...... 3 3 Head and mesosoma green to bluish; facial tomentum mostly limited to paraocular areas (Southern Ontario and Eastern USA north of Florida)...... L. tegulare - Head and mesosoma blue; facial tomentum more evenly distributed on face (Southeastern USA) ..... L. puteulanum Discussion DNA barcodes are increasingly becoming a standard tool used by taxonomists (e.g. Van Nieukerken 2007; Pyle et al. 2008; Gibbs in press; Gibbs & Sheffield in press) but the method continues to be criticized in the literature (Rubinoff et al, 2007; Wheeler 2008). Some potential drawbacks were dismissed above for the data presented herein. The remaining criticisms of relevance to the present study are primarily i) DNA barcoding data are presented in a phenetic manner using NJ trees, ii) a short sequence of mtDNA is insufficient for the detection of new species and iii) species delimitation using % sequence divergence is arbitrary. I address these issues in turn. 32 · Zootaxa 2032 © 2009 Magnolia Press GIBBS FIGURE 14. Dorsal views of male T2 showing punctation in (A) L. ellisiae and (B) L. puteulanum. i) The use of a neighbour-joining algorithm to provide specimen identification ignores several decades of study that demonstrate the superiority of phylogenetic over phenetic philosophical criteria (Farris 1977, 1983; Hull 1988). Nevertheless, the phenetic approach is seemingly necessitated by the rapidity of NJ algorithms combined with the near impossibility of performing a rigorous phylogenetic analysis with the large number of short sequences commonly generated in DNA barcoding studies. Here I have demonstrated that rigorous phylogenetic analysis can be performed to permit diagnosis of cryptic species as both monophyletic and diagnosable entities. The latter based upon discrete characters (nucleotide substitutions), which are an improvement over phenetic distances (DeSalle et al. 2005; DeSalle 2005). Consequently, the rigour of LASIOGLOSSUM (DIALICTUS) TEGULARE SPECIES GROUP Zootaxa 2032 © 2009 Magnolia Press · 33 cladistic methodology can be applied to DNA barcode data when the question being addressed is of manageable size. The application of more rigorous phylogenetic analyses to DNA barcode data is becoming more frequent in the literature (Skevington et al. 2007; Hastings et al. 2008; Witt et al. 2008). ii) It has been known for a long time that there are numerous species that cannot be differentiated solely on the basis of traditional morphological analysis (see Avise 2004). An example from the bees is Halictus ligatus, an abundant and easily recognised “species” until demonstrated to be two well differentiated species using allozymes and DNA sequence data (Carman & Packer 1997; Danforth et al. 1998, 1999). One of these cryptic species, H. poeyi, is found to the southeast of the Appalachian mountains, the other, H. ligatus, to the northwest and they are sympatric in the Piedmont region where the very high differentiation found at nuclear loci as well as mitochondrial sequence divergence is entirely maintained (Packer 1999). Despite large levels of genetic differentiation, the two species have remained resistant to morphological diagnosis. Even morphometric analysis of wing venation and male genitalia has failed to result in discrimination (Packer, unpublished data). In the results for the L. (D.) tegulare species complex presented here, morphological diagnosis is possible for all of the species found to be differentiated genetically. Morphological differences between these species could be (and for a long time was) mistaken for intraspecific variation without an independent dataset to test species limits (DeSalle et al. 2005). Consequently, a short sequence of mtDNA is useful for species discovery (e.g. Hebert et al. 2004) and can provide data of use for integrative taxonomy (Smith et al. 2007; Miller 2007; Padial & De La Riva 2007). iii) Sequence divergence is an easy metric to measure but not necessarily an easy variable to interpret. While many cases of completely non-overlapping % differences between intraspecific and interspecific divergences are known, the mere fact of evolution will result in numerous instances of overlap between them (e.g. Meyer & Paulay 2005). Precisely the same is true of morphological analysis: usually there are sufficient differences between sister species to permit identification (though in many insects this remains possible for only one sex) but there are many cases in which differentiation is possible only through additional information, such as DNA sequence or ecological data (e.g. Smith et al. 2008). While there are rare cases of zero DNA barcode differentiation, there are many examples of zero, or almost zero, morphological differentiation between diagnosable species (Avise 2004; see also Packer et al. in press). In this study, I used DNA barcode data to discover genetically discrete groups of individuals for which I could then find discrete morphological differences. The level of morphological differentiation among the newly discovered or resurrected species is sometimes meagre. Nonetheless, such limited amounts of differentiation are commonly found among species in the Halictidae (Wheeler 1928; Michener 2007), as well as in many other insects (Hebert et al. 2004; Smith et al. 2006, 2008) and as noted above, genetically well-differentiated species are sometimes morphologically indistinguishable. The issue of arbitrary levels of differentiation for the separation of organisms at the species level is just as much a problem for traditional morphological approaches as it is for DNA barcoding. The only difference is that the quantitative aspect of DNA barcoding makes the arbitrariness more obvious. Nonetheless, when individuals fall into clearly discrete clusters on an NJ tree with interspecific differentiation clearly greater than intraspecific differentiation and these clusters are robust to phylogenetic analysis, it is not unreasonable to refer to the clusters as representing species. In the cases presented here, morphological differences discriminate clades that are also diagnosable with the mtDNA sequence differences and are of similar magnitude to that found among uncontroversial species elsewhere among the bees. Acknowledgements My sincere appreciation is given to Dr. Laurence Packer for his helpful advice and supervision regarding this project and for commenting on an earlier version of the manuscript. The ANSP, CUIC, INHS, KUNMH, NMNH, Dr. Terry Griswold of Utah State University, Sam Droege of the USGS Patuxent Wildlife Research Center, Dr. Julio Genaro, and many others provided specimens that made this research possible. Dr. Chris 34 · Zootaxa 2032 © 2009 Magnolia Press GIBBS Darling generously allowed me access to imaging equipment at the Royal Ontario Museum. Claudia Ratti helped prepare the figures for which I am grateful. This research was supported through funding to the Canadian Barcode of Life Network from Genome Canada, NSERC, and other sponsors listed at www.BOLNET.ca. Ontario Graduate Scholarships in Science and Technology awarded to the author are greatly appreciated. Specimens collected in Great Smoky Mountain National Park contributed to this work and were sampled as part of the National Park Service study number GRSM-00430. Literature cited Ascher, J. S. et al. (2008) Apoidea species guide. Available from http://www.discoverlife.org/mp/ 20q?guide=Apoidea_species (accessed 31 December 2008) Avise, J.C. (2000) Phylogeography: the History and Formation of Species. Harvard University Press, Boston, Massachusetts, viii + 447 pp. Avise, J.C. (2004) Molecular Markers, Natural History and Evolution. Springer, New York, New York, 684 pp. Brady, S.G., Sipes, S., Pearson A. & Danforth, B.N. (2006) Recent and simultaneous origins of eusociality in halictid bees. 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